The induction of the nervous systeim one of the earliest and most dramatic events of vertebrate development, has challenged and frustrated embryologists since the organizer graft experiments of Spemann and Mangold. Classical work established that the gastrula stage ectodeim of amphibian and other vertebrate embryos gives rise to the neural plate in response to signals from the adjacent dorsal mesodeii (Spemaim's orgarizer). In the absence of this influence, as on the ventral side or in explants made before gastrulation, the ectodeim differentiates only as epiderims. Thus, development as epidermiis was generally assumed to be a fall-back, or default fate for the gastl-ula ectodeim requiring no cell-cell comunication, while neural specification was contingent on receipt of signals. However, much effort over several decades failed to identify the chemical substances responsible for neural induction in the embryo, though a variety of rather curious materials were found to be able to neutralize salamander ectoderm. Recent studies of the amphibian embryo have identified three diffusible factors with neural inducing ability: noggin (Lamb et al., 1993), follistatin (Hernati-Brivailou et al., 1994) and chordin (short gastrulation) (Sasai et al., 1995; Sasai et al., 1994). All three factors mimic the signal(s) which emanates from the organizer and converts ventral ectoderm (epidermis) to dorsal ectoderm (nervous tissue).
Other recent work has led to a second promising molecular candidate for a neural inducing signal and at the same time suggested a new twist on the long-held classical model of neural and epiderrnal specification. First, Gruz and others revealed that Xenopus ectoderm cultured during early gastrula stages as a dispersed cell population formed neural tissue even though it receives no signals from the mesodenn during this period. More recently, Brivanlou and Melton discovered that injection of a dominanit-negative fonn of the activin receptor could neuralize ectodennal explants, again in the apparent absence of mesodenm Finally, the activin antagonist follistatin could also cause neural differentiation. These fmdings led Brivaiilou and Melton to propose that the cells of the early gastrula animal cap are disposed to form neural tissue, in the absence of further influences. In this sense one could speak of a default neural fate for the ectoderm The neural "default model" of neural induction argues that the differentiation of epidermis requires inductive signals, while the neuralization of the dorsal ectoderm requires only an inhibition of this signaling.
Epidermal specification, and thus the inhibition of neural fate, results from cell-cell commuiication within the prospective ectoderm When this signaling is interrupted, by dispersing the cells or by molecular antagollists, neural tissue forms. Neural induction by the dorsal mesodeln, in this model, would work in the same way, that is by blocking epidermalizing signaling within the animal cap. Since both the truncated activin receptor and follistatin could accomplish this, activin seemed likely to be the factor that mediated epidermal specification. The further discovery that follistatin was expressed in the organizer region in Xenopus, firom where it could act to block activin signaling in the dorsal ectodeim and thus permit neural tissue to form, naturally suggested follistatin as an endogenous neural inducer. Although these were enticing speculations, there was no direct evidence that activin could act to specify epidermis.
More recently it has been disclosed that BMP-4, may be the endogenous neural inhibitor and epidermal inducer (see, U.S. patent application Ser. No. 08/413,047, filed Mar. 29, 1995, and U.S. patent application Ser. No. 08/622,860, filed Mar. 29, 1996 hereby incorporated herein in their entireties). Ectodermal ("animal cap") explants form epidermis when cultured intact; these explants will neuralize if subjected to prolonged dissociation [Grunz and Tacke, Cell Diff Devl., 28:211 (1989); Sato and Sargent, Dev. Biol., 134:263 (1989); Godsave and Slack, Dev. Biol., 134:486 (1989)]. Soluble BMP-4 can induce epidermis in dissociated ectoderm, substituting for the epidennal inducer presumably lost by dilution [Wilson and Hermati-Brivanlou, Nature, 376:331 (1995)].
BMP-4 is a member of a set of closely related proteins that form a subgroup witlihi the larger TGF-.beta. superfamily of secreted growth factors that also includes activin and the TGF's proper. First purified firom bone as activities capable of promoting bone regrowth, the BMPs have more recently been found in early vertebrate embryos, where they appear to play a variety of roles. In Xenopus, several groups have shown that BMP-2 and BMP-4 are capable of inducing ventral mesoderm, as well as ventralizing mesoderm induced by activin. Recent work showing that BMP-4 is expressed in the ventral marginal zone, and that a dominant negative version of a BMP receptor has strong dorsalizing effects on early embryos lends further support to the idea that BMP-4 acts in vivo to ventralize the marginal zone. However, little is known with regard to factors that are involved in epidermal induction downstream in the BMP-4 signaling pathway. Therefore, there is a need to identify such factors in order to identify agents that can stimulate epidermal growth.
Protein translation rates increase in response to a variety of peptide growth factors [Rhoads, Curn. Opin. Cell Biol., 3:1019 (1991); Frederickson and Sonenberg, in Translational Regulation of Gene Expression, J. Illan, Ed., Plenum Press, New York, pp. 143-162 (1993)]. In several studies, soluble factors have been shown to enhance translation through the modification of cytoplasmic proteins involved in translation initiation [Sonenberg, in Translational Control, J. W. B. Hershey, M. B. Matthews, and N. Sonenberg, Eds., Cold Spring Harbor Press, New York, pp. 245-269 (1996)]. Interestingly, stimulation of the initiation machinery does not always result in a general increase in the rate of translation: various growth factor treatments can dramatically elevate the translation rates of specific mRNAs [Sonenberg, 1996, supra]. Enhaniced activity of translation initiation factors may preferentially lead to the expression of mRNAs with a complex secondary structure in their 5' untranslated regions (UTR) [Sonenberg, 1996, supra; Brown and Schreiber, Cell, 86:517 (1996)]. This selective translation can have profound consequences for cell fate. For example, the overexpression of translation initiation factor eIF-4E in Xenopus embryos induces mesoderrn in cells that would otherwise develop as epidermis [Klein and Melton, Science, 265:803 (1994)]. Ectopic eIF-4E preferentially elevates the translation of mRNA encoding activin, a mesoderm-inducing growth factor.
eIF-4E, along with eIF-4A and eIF-4G, form the multisubunit cap-binding complex eIF-4F. This initiation complex is thought to unwind secondary structure in the 5' UTR of mRNA to allow ribosome binding and thus initiate translation [Sonenberg, 1996, supra]. The helicase activity of eIF-4F is thought to be conferred by the eIF-4A subunit, in conjunction with eIF-4B [Rozen et al., Mol. Cell. Biol., 10:1134 (1990); Pause et al., EMBO J., 13:1205 (1994)]. Although other RNA helicases have been implicated in specific interactions with MRNA [Lasko and Ashbumer, Nature, 335:611 (1988); Hay et al., Cell, 55:577 (1988); Liang et al., Development, 120:1201 (1994)], no evidence has thus far demonstrated a role for the helicase component of the initiation complex in binding specificity.
There is presently a great need for generating neural and epideimal tissue for a variety of medical treatments. Manipulating cellular factors that are part of the pathways involved in the induction of either epideimal or neural cellular differentiation is a method likely to be successful for producing such tissues. Any of these factors along the pathway could conceivably be used in the induction of epideimal or neural cellular differentiation, or as targets for drug screens that are employed to identify molecules that will stimulate or inhibit the induction of epidermal or neural cellular differentiation.
The citation of any reference herein should not be construed as an adlmission that such reference is available as "Prior Art" to the instant application.